Lactam nucleic acids

ABSTRACT

Novel β-lactam monomers bearing various functional groups are prepared. The novel β-lactam monomers can be joined into oligomeric compounds such as via preferred phosphate linkages including phosphodiester and phosphorothioate linkages. Useful functional groups include nucleobases as well as polar groups, hydrophobic groups, ionic groups, aromatic groups and/or groups that participate in hydrogen bonding. The oligomeric compounds are useful as diagnostic and research reagents.

This is a division, of application Ser. No. 08/243,368, filed May 16,1994, now U.S. Pat. No. 5,554,746, the disclosure of which is hereinincorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to a new class of polymeric compoundsfor binding to complementary DNA and RNA strands. In particular, theinvention concerns compounds wherein naturally-occurring nucleobases orother nucleobase-binding moieties are covalently bound to anoligolactamtide, i.e., a backbone comprising repeating units that arephosphoric acid esters of β-lactams. These compounds are useful fordiagnostics, research reagents and therapeutics. The present inventionis also directed to processes for synthesizing such compounds, and tointermediates used in such processes.

The ultimate mechanism of action for most conventional therapeuticagents, i.e. drugs, is by way of modulation of one or more targetedendogenous proteins, e.g., enzymes. Such agents, however, typically lacktotal specificity for their target proteins, and instead interact withother proteins as well. Thus, a relatively large dose of the agent mustbe used to effectively modulate a target protein; and the agent cancause undesired side effects as the result of interference in the actionof the non-target proteins. Typical daily doses of such conventionalagents are from 10⁻⁵ -10⁻¹ millimoles per kilogram of body weight or10⁻³ -10 millimoles for a 100 kilogram person. If this modulationinstead could be effected by interaction with or inactivation at a pointin the biological pathway at an earlier stage, a dramatic reduction inthe necessary amount of the therapeutic agent necessary could likely beachieved, along with a corresponding reduction in side effects. Furtherreductions could be effected if such interaction could be renderedsite-specific.

Proteins are produced in biological systems ultimately from genes thatencode them. A gene performs its basic function by transcription of itsencoded information to messenger RNA (mRNA) which, by interaction withthe ribosomal complex in a synthetic process called translation, directsthe assembly of the protein coded for by its sequence. Translationrequires the presence of various co-factors and the amino acid buildingblocks for the protein, and their transfer RNAs (tRNA), all of which arepresent in normal cells.

In order for transcription to be initiated, there must be recognition ofa specific promoter DNA sequence by the RNA-synthesizing enzyme, RNApolymerase. In many cases in prokaryotic cells, and probably in allcases in eukaryotic cells, this recognition is preceded bysequence-specific binding of a protein transcription factor to thepromoter. Other proteins which bind to the promoter, but whose bindingprohibits action of RNA polymerase, are known as repressors. Thus, geneactivation typically is regulated positively by transcription factorsand negatively by repressors. These genetic regulatory mechanisms andthe ability to influence them have significant implications fordiagnostics and therapeutics. Since a functioning gene continuallyproduces mRNA, it is advantageous if gene transcription is modulated orblocked totally.

Oligonucleotides have been shown to interact with mRNA and othercomponents associated with gene transcription. By virture of suchinteraction, synthetic preparations of naturally occurringoligonucleotides and synthetic oligonucleotide derivatives and analogswhich do not occur in nature, have become valuable tools for researchand important agents in diagnostic, therapeutic and other applications.Increasingly, there is a demand for such improved oligonucleotides,oligonucleotide analogs, as well as for methods for their preparation.

Oligonucleotides have been used in a number of areas of research. Ingenomic research oligonucleotides have been used as probes and primers.Oligonucleotides are also useful in devising diagnostics since they canspecifically hybridize to nucleic acids of interest in the etiology of agiven disease. Oligonucleotides are also being tested as therapeuticmoieties in the treatment of disease states in animals and man. Forexample, workers in the field have now identified oligonucleotidetherapeutic compositions that are capable of modulating expression ofgenes implicated in viral, fungal and metabolic diseases. It has nowbecome routine to synthesize oligodeoxyribonucleotides andoligoribonucleotides having hundreds of base pairs (bp) by solid phasemethods using commercially available, fully automatic synthesismachines. In short, oligonucleotides are important molecules having alarge commercial impact in biotechnology and medicine. As a consequence,improved oligonucleotides and methods for the synthesis of improvedoligonucleotides, which afford reduced cost and environmental impact,along with increased efficiency and convenience, are also in demand.

Unmodified oligonucleotides, i.e. natural phosphodiester linkedoligonucleotides, are unpractical for many uses because they have shortin vivo half-lives or they suffer from a limited ability to penetratecell membranes. In order to improve half life as well as membranepenetration, a large number of variations in polynucleotide backboneshave been undertaken. These variations include the use ofmethylphosphonates, monothiophosphates, dithiophosphates,phosphoramidates, phosphate esters, bridged phosphoramidates, bridgedphosphorothioates, bridged methylenephosphonates, dephosphointernucleotide analogs with siloxane bridges, carbonate bridges,carboxymethyl ester bridges, acetamide bridges, carbamate bridges,thioether, sulfoxy, sulfono bridges, various "plastic" DNAs, α-anomericbridges, and borane derivatives. These analogues have variousproperties. Only a few have proved to have such a combination ofproperties that render them substitutes for natural oligonucleotides.

One of the most useful oligonucleotide analogues discovered to date is aclass of compounds known as peptide nucleic acids. These compounds havebeen found to have enhanced hybridization to complimentary DNA and RNAstrands as compared to most other known oligonucleotide analogues aswell as nuclease and protease stability. Peptide nucleic acids haveneutral, amide linked backbones. Retention of a phosphorous atom inoligomeric backbones is considered to be highly desirable for certainutilities since such backbones are capable of providing a chargedspecies and for providing a potential site for interactions withpeptides, as for instance with transcription factors.

The β-lactam nucleic acid oligomers of the present invention areexpected to exhibit superior properties as compared to prior reagents inthat they mimic the higher affinity for complementary single strandedDNA (ssDNA) exhibited by peptide nucleic acids but, unlike peptidenucleic acids, the β-lactam backbone is phosphorous linked resulting ina charged compound. The β-lactam compounds are also expected to formtriple helices where a first β-lactam nucleic acid strand binds with RNAor ssDNA and a second β-lactam nucleic acid strand binds with theresulting double helix or with the first β-lactam nucleic acid strand.β-lactam nucleic acids are generally water soluble to facilitate bothdiagnostic and research reagent use as well as cellular uptake.Moreover, β-lactam nucleic acids contain the β-lactam structure. Suchstructure is expected to make them biostable and resistant to enzymaticdegradation, for example, by proteases.

With regard to the novel methods of preparation for the β-lactam nucleicacids of the present invention, it is noted that methods have beenemployed heretofore for preparing oligonucleotides that utilizesolid-phase synthesis wherein an oligonucleotide is prepared on apolymer or other solid support. Such solid-phase synthesis relies onsequential addition of nucleotides to one end of a growingoligonucleotide. Typically, a first nucleoside is attached to anappropriate support, e.g. long chain alkyl amine controlled pore glass(LCAA CPG), and nucleotide precursors are added stepwise to elongate thegrowing oligonucleotide. The nucleotide precursors are conventionallyreacted with the growing oligonucleotide using the principles of a"fluidized bed" for mixing of the reagents, where solid supportscomposed of silica are used. While some of the techniques of suchconventional processes are used in the novel methods of the presentinvention, nowhere is there a suggestion of the novel reactants andsteps that characterize the present methods.

OBJECTS OF THE INVENTION

It is an object of this invention to provide novel monomericβ-lactamsides and β-lactamtides.

It is another object of the invention to provide novel oligomericβ-lactamtides.

It is a further object of the invention to provide methods for preparingand using oligomeric β-lactamtides.

SUMMARY OF THE INVENTION

Compounds of the invention may include oligomeric compounds of StructureI: ##STR1## wherein B₁ and each B_(m), independently are a naturallyoccurring nucleobase, a non-naturally occurring nucleobase, a DNAintercalator, a nucleobase-binding group, hydrogen, hydroxyl, a (C₁-C₄)alkanoyl, an aromatic moiety, or a heterocyclic moiety, which may beoptionally substituted with one or more additional functional groupsselected from hydrogen, hydroxyl, alkyl, substituted alkyl, alkaryl oraralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino orsubstituted silyl, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligolactamtide, a group for improvingthe pharmacodynamic properties of an oligolactamtide, or a reporterligand;

A₁ and each A_(m), independently are (CR₆ R₇)_(x) where R₆ and R₇ areindependently selected from the group consisting of hydrogen, (C₂-C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C₁ -C₆)alkoxy, (C₁-C₆)alkylthio, NR₃ R₄ and SR₅, where each of R₃ and R₄ is independentlyselected from the group consisting of hydrogen, (C₁ -C₄)alkyl, alkoxy,or alkylthio-substituted (C₁ -C₄)alkyl, alkoxy, alkylthio and amino; andR₅ is hydrogen, (C₁ -C₆)alkyl, hydroxy-, alkoxy-, oralkylthio-substituted (C₁ -C₆)alkyl, or R₆ and R₇ taken togethercomplete an alicyclic system;

E₁ and E₂, independently, are H, a hydroxyl protecting group, anactivated solid support, a conjugate group, a reporter group, apolyethylene glycol, an alkyl, an oligonucleotide, a phosphate, aphosphite, an activated phosphate, or an activated phosphite;

Z is OH, SH, CH₃, or NR₁ R₂ ;

R₁ and each R₂, independently, are H, C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, a heterocycle, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, or C₇ -C₁₄ aralkyl;

Y is oxygen or sulfur;

n is an integer from 1 to 60;

e₁ and each e_(m), independently are 0 or an integer from 1 to 6;

b₁ and each b_(m), independently are 0 or an integer from 1 to 6;

In certain preferred embodiments, B₁ and each B_(m) are independentlyselected as naturally occurring nucleobases. In further preferredembodiments B₁ and each B_(m) are independently selected as nonnaturally occurring nucleobases. In yet other preferred embodiments e₁,e_(m), b₁, and each b_(m) are, independently, an integer from 1 to about4.

In preferred embodiments of the invention n is from 1 to about 40. Amore preferred range of n is from 1 to about 20.

Compounds of the invention may also include monomeric compounds ofStructure II: ##STR2## wherein B_(m) is a naturally occurringnucleobase, a non-naturally occurring nucleobase, a DNA intercalator, anucleobase-binding group, hydrogen, hydroxyl, a (C₁ -C₄)alkanoyl, anaromatic moiety, or a heterocyclic moiety, which may be optionallysubstituted with one or more additional functional groups selected fromhydrogen, hydroxyl, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl,Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino or substitutedsilyl, an RNA cleaving group, a group for improving the pharmacokineticproperties of an oligolactamtide, a group for improving thepharmacodynamic properties of an oligolactamtide, or a reporter ligand;

A₁ is (CR₆ R₇)_(x) where R₆ and R₇ are independently selected from thegroup consisting of hydrogen, (C₂ -C₆)alkyl, aryl, aralkyl, heteroaryl,hydroxy, (C₁ -C₆)alkoxy, (C₁ -C₆)alkylthio, NR₃ R₄ and SR₅, where eachof R₃ and R₄ is independently selected from the group consisting ofhydrogen, (C₁ -C₄)alkyl, alkoxy, or alkylthio-substituted (C₁ -C₄)alkyl,alkoxy, alkylthio and amino; and R₅ is hydrogen, (C₁ -C₆)alkyl,hydroxy-, alkoxy-, or alkylthio-substituted (C₁ -C₆)alkyl, or R₆ and R₇taken together complete an alicyclic system;

X is an integer from 1 to 10, and can be 0 only when B_(m) is nothydrogen or hydroxyl;

E1 and E2, independently, are H, a hydroxyl protecting group, anactivated solid support, a conjugate group, a reporter group, apolyethylene glycol, an alkyl, an oligonucleotide, a phosphate, aphosphite, an activated phosphate, or an activated phosphite;

e_(m) is 0 or an integer from 1 to 6;

b_(m) is 0 or an integer from 1 to 6;

In certain preferred embodiments, B_(m) is selected as a naturallyoccurring or non naturally occurring nucleobase.

In other preferred embodiments e_(m) and b_(m) are independently aninteger from 1 to about 4.

Further in accordance with this invention there are provided methods forpreparing β-lactamtides that include the steps of:

Providing a β-lactam having a functional group, a protected hydroxylgroup, and a hydroxyl group covalently bonded thereto. The functionalgroup is a naturally occurring nucleobase, a non-naturally occurringnucleobase, a DNA intercalator, a nucleobase-binding group, hydrogen,hydroxyl, a (C₁ -C₄)alkanoyl, an aromatic moiety, or a heterocyclicmoiety, furthermore, said functional group can be optionally substitutedwith one or more additional functional groups selected from hydrogen,hydroxyl, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN,CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino or substitutedsilyl, an RNA cleaving group, a group for improving the pharmacokineticproperties of an oligolactamtide, a group for improving thepharmacodynamic properties of an oligolactamtide, or a reporter ligand.Attaching the β-lactam to a solid support via the hydroxyl group.Treating the protected hydroxyl group with a deprotecting reagent togive a free hydroxyl group. Reacting the free hydroxyl group with aβ-lactamtide having an activated phosphite group, a functional group asdefined above, and a protected hydroxyl group thereon to form aphosphite triester. Oxidizing the phosphite triester to a phosphatetriester.

Repetition of the above steps of deprotecting the protected hydroxyl andtreating with a β-lactamtide adds a further β-lactamtide phosphitetriester unit. Oxidation of the resulting β-lactamtide phosphitetriester unit will give the phosphate triester. This iterative processthus increases the length of the oligomer.

In certain embodiments of the invention the functional group attached tothe β-lactam and the functional group attached to the β-lactamtide isattached using a tether. The tether is selected from (CR₆ R₇)_(x) whereR₆ and R₇ are independently selected from the group consisting ofhydrogen, (C₂ -C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C₁-C₆)alkoxy, (C₆ -C₆)alkylthio, NR₃ R₄ and SR₅, where each of R₃ and R₄is independently selected from the group consisting of hydrogen, (C₁-C₄)alkyl, alkoxy, or alkylthio-substituted (C₁ -C₄)alkyl, alkoxy,alkylthio and amino; and R₅ is hydrogen, (C₁ -C₆)alkyl, hydroxy-,alkoxy-, or alkylthio- substituted (C₁ -C₆)alkyl, or R₆ and R₇ takentogether complete an alicyclic system, and where _(x) is an integer from1 to 10, and can be 0 only when said functional group is not hydrogen orhydroxyl. In other embodiments of the invention the hydroxyl group andthe protected hydroxyl group attached to the β-lactam and the protectedhydroxyl group and the activated phosphite group attached to theβ-lactamtide are attached using a C₁ to about C₆ alkyl tether. A morepreferred length of the alkyl tether is C₁ to C₃ alkyl tether.

In certain further embodiments of the invention the β-lactam issubstituted with the hydroxyl group covalently bonded to the N-1position and the protected hydroxyl covalently bonded to the C-4position. In other embodiments the hydroxyl group is covalently bondedto the N-1 position and the protected hydroxyl group is covalentlybonded to the C-4 position are bound via tethers that are from C₁ toabout C₃ alkyl. In certain further embodiments of the invention theβ-lactamtide is substituted with the protected hydroxyl group covalentlybonded to the C-4 position and the activated phosphite group covalentlybonded to the N-1 position. In other embodiments the protected hydroxylgroup covalently bonded to the C-4 position and the protected hydroxylgroup covalently bonded to the N-1 position are bound via tethers thatare from about C₁ to about C₃ alkyl.

In practicing certain aspects of the invention a β-lactam having a firstand a second hydroxyl group can be formed by treatment of a fullyprotected dihydroxy substituted β-lactam with a weak base to selectivelycleave the protecting group from the hydroxyl bonded to the N-1position. The phosphite triester can be treated with a capping reagentprior to oxidation. The phosphite triester can be oxidized to aphosphate triester or to a phosphorothioate.

DESCRIPTION OF PREFERRED EMBODIMENTS

The monomeric compounds of the invention each include a β-lactam moietythat is substituted at the N-1, C-3, and C-4 positions with tethered oruntethered functional groups as shown in formula I and II. Preferredsubstitutions at the C-3 position can include naturally occurringnucleobases e.g. thymine, adenine, cytosine, guanine, and uracil as wellas non naturally occurring nucleobases and other functional moieties.Substitutions at the C-3 position can also include an optional A_(m)group. The B_(m) group serves as a functional moiety. The A_(m) groupcan serve as a functional moiety or it can serve as a tether for theB_(m) group. Hydroxyl groups are included at the N-1 and C-4 positionseither with or without alkyl groups that act as tethers or spacespanning groups. These hydroxyls can mimic the 3' and 5' hydroxyls of adeoxyribonucleotide in that they can be used to link the β-lactam ringsin a manner analogous to nucleotides. They can be linked to solidsupport, other β-lactams through phosphate linking moieties, or to othergroups as is practiced in the synthesis of conventionaloligonucleotides.

The use of protecting groups of varying reactivities for the hydroxylsattached directly or indirectly to the C-4 and the N-1 position and forgroups on the B_(m) and or A_(m) groups attached to the C-3 positionduring the synthesis, enables selective deprotection. Thus, the hydroxylgroup at the N-1 position can be protected allowing it to be selectivelycleaved such as by using a fluoride source such as HF/pyridine for a R₃Si protecting group, or KCN in the case of an acetyl protecting group,leaving the functional groups at the C-3 and C-4 position protected. Theresulting free hydroxyl group at the N-1 position may be further reactedwith an activated solid support or functionalized to thephosphoramidite. As will be evident to those skilled in the art a numberof oligomeric compounds can be synthesized using these synthons.

The β-lactam protected phosphoramidite e.g. a DMT-phosphoramidite, isused analogously to the DMT-phosphoramidite of a deoxyribonucleotidemaking the present invention amenable to all the chemistries used in theassembly of oligodeoxynucleotides e.g. phosphotriester, H-phosphonate,and others.

The present invention presents novel β-lactamsides, β-lactamtides, andoligo-β-lactamtides e.g. β-lactam nucleic acids. The oligo-β-lactamtidescan be prepared entirely of β-lactam monomers or they can be prepared toinclude other monomeric units e.g. ribonucleotides and analogs thereofor peptide nucleic acids and analogs thereof wherein at least one of themonomers of the resulting oligomeric composition is a β-lactam monomerof the invention.

Synthesis of the β-lactamsides, β-lactamtides, and oligo-β-lactamtidesis preferably by a stepwise procedure wherein intermediates are preparedand reacted sequentially with each other. The procedures described belowrepresent certain preferred embodiments and correspond to the followingschemes. ##STR3##

An alkyl diol, 1 is protected using an R₃ Si-- type protecting groupe.g. TBDMS, to give the monoprotected diol, 2. The monoprotected diol isoxidized to the aldehyde, 3 via e.g. a Swern oxidation. In a separatesynthesis an alkyl hydroxyl amine, 4 is protected with a weak baselabile protecting group e.g. acetyl, to give, 5 which is further reactedwith the aldehyde, 3 to give a diprotected imine, 6 or an oxime whenb_(m) =0 e.g. there is no (CH₂) between N and OP_(a) in the imine, 6. Abromoalkylmethyl ester is optionally reacted with an A_(m), 8 asdescribed above, followed by reaction with B_(m), 9 (e.g. a nucleobase,a modified nucleobase, or other group as discussed above) that isprotected if necessary, to give a B-A-alkyl carboxy compound, 10 whereinA is optional. The B-A-alkyl carboxy compound is further reacted withthe diprotected imine or oxime, 6 to give a substituted β-lactam, 11through an acid chloride imine or oxime condensation or by ketene oximecondensation. The substituted β-lactam is converted into thephosphoramidite, 13 by first selectively removing the P_(c) protectinggroup e.g. TBDMS, which can be selectively removed by a fluoride sourceleaving other acid labile and base labile protecting groups used in thesynthesis unaffected and then reacting the resultant free hydroxyl withan acid labile protecting group used in standard oligodeoxynucleotidesynthesis e.g. dimethoxytrityl, to give the trityl protected β-lactam,12. Removal of the P_(a) protecting group with a nucleophilic group suchas cyanide ion followed by reaction with a standard amidite reagent like(β-cyanoethoxy)-chloro-N,N-diisopropylamino)phosphine gives thephosphoramidite of the substituted-β-lactam.

It can readily be seen that by altering the starting materials a largenumber of diverse structures can be synthesized. The use of differentA_(m) groups will modify the distance from the β-lactam backbone to theBase moiety (B_(m)). When A_(m) is not incorporated as a separateentity, it may still be incorporated by altering the ester, 7. A smallalkyl chain can be incorporated as for instance using methylbromoproprionate to give A_(m) equal to CH₂ or using methylbromobutyrate as the ester to give A_(m) equal to C₂ H₄. Altering thenumber of alkyl spacing groups e.g. e_(m) and b_(m), will effect theoverall length of the final oligomer. As illustrated in the belowexamples, there are numerous reagents available with protecting groupse.g. benzyl amines and benzyl esters that may be used in the synthesiswithout a prior protecting step,. Holding the e_(m), b_(m), and A_(m)groups constant and altering the base moiety (B_(m)) used in thesynthesis will also provide a large number of diverse compounds.

Lactam Nucleic Acids as Oligomers

The compounds of the present invention may also be described asoligomers wherein at least one of the subunits thereof is a β-lactamnucleic acid of the structure: ##STR4## wherein A_(m), B_(m), E₁, E₂,e_(m), and b_(m) have the same meaning as defined above. The term"subunits" as used herein, refers to β-lactam nucleic acid moieties thatcan form polymers and to naturally occurring nucleotides and nucleotideanalogs as well as other commonly used monomers that are routinely usedfor preparing oligomeric sturctures using standard methods andtechniques. Such repeating subunits form polymers referred to as"oligomers", and this term, as used herein, may refer to oligomers inwhich substantially all subunits of the oligomer are β-lactam subunitsas illustrated above. Oligomers of the present invention may alsocomprise one or more subunits that are not β-lactam subunits e.g.naturally occurring nucleotides, nucleotide analogs, or other commonlyused monomers as long as at least one subunit is a β-lactam nucleicacid. Thus, oligomers as used herein may refer to a range of oligomersfrom oligomers having only one β-lactam nucleic acid subunit tooligomers in which every subunit is a β-lactam nucleic acid subunit.

Those subunits which are not β-lactam nucleic acid subunits comprisenaturally occurring bases, sugars, and intersugar (backbone) linkages aswell as non-naturally occurring portions. Sequences of oligomers of thepresent invention are defined by reference to the B group (for β-lactamnucleic acid subunits) or nucleobase (for nucleotide subunits) at agiven position. Thus, for a given oligomer, the nomenclature is modeledafter traditional nucleotide nomenclature, identifying each β-lactamnucleic acid subunit by the identity of its B group, such as theheterocycles adenine (A), thymine (T), guanine (G) and cytosine (C) andidentifying nucleotides or nucleosides by these same heterocyclesresiding on the sugar backbone. The sequences are conveniently providedin traditional 5' to 3' direction.

Oligomers of the present invention may range in length from about 2 toabout 60 subunits. In other embodiments of the present invention,oligomers may range in length from about 2 to about 40 subunits. Instill other embodiments of the present invention oligomers may range inlength from about 2 to about 20 subunits. In yet further embodiments ofthe present invention, oligomers may range in size from about 6 to about18 subunits in length.

β-Lactam phosphoramidites of structure I are used to form oligomericstructures of the invention wherein, the A_(m), B_(m), e_(m), and b_(m),are as described above and E₁ is an activated phosphite and E₂ is anacid labile hydroxyl protecting group e.g. dimethoxytrityl chloride. Thesubunits or phosphoramidites of the invention are covalently linked withphosphate linkages using standard solid and solution phase chemistries.Representative solution phase techniques are described in U.S. Pat. No.5,210,264, issued May 11, 1993 and commonly assigned with thisinvention. Representative solid phase techniques are those typicallyemployed for DNA and RNA synthesis utilizing standard phosphoramiditechemistry. (see, e.g., Protocols For Oligonucleotides And Analogs,Agrawal, S., ed., Humana Press, Totowa, N.J., 1993.) A preferredsynthetic solid phase synthesis utilizes phosphoramidites as activatedphosphites. The phosphoramidites utilize P^(III) chemistry. Theintermediate phosphite compounds are subsequently oxidized to the P^(V)state using known methods. This permits synthesis of the preferredphosphodiester or phosphorothioate phosphate linkages depending uponoxidation conditions selected. Other phosphate linkages that can beprepared include phosphorodithioates, phosphotriesters, alkylphosphonates, phosphoroselenates and phosphoramidates.

One preferred synthesis of β-lactam nucleic acids of the presentinvention starts with attaching a β-lactam to a solid support carrierusing the N-1-hydroxyl group which may include an alkyl tether.Alternatively it is possible to attach the β-lactam to the syntheticresin solid support through formation of a phosphite triester or otherlinkage. Thus, the β-lactam is reacted in a manner analogous tonucleosides having 3'--OH and 5'--OH groups, which are used in preparingpolynucleotides, as described in, e.g., U.S. Pat. Nos. 4,458,066;4,500,707; and 5,132,418. The 3'--OH group of the nucleoside correspondsto the 1-N-hydroxyl group of the β-lactam, while the 5'--OH groupcorresponds to the hydroxyl group attached to the 4-position of theβ-lactam. These hydroxyls are attached to the β-lactam either with orwithout an alkyl tethering group. In a manner analogous to the knownmethods for solid support synthesis of polynucleotides, the dihydroxyβ-lactam is covalently coupled to the synthetic resin solid supportusing a conventional coupling agent.

The nature of the reactive group which bonds the dihydroxy β-lactam tothe synthetic resin support is not critical, but should preferably bereadily hydrolyzable in order to permit separation of the final β-lactamnucleic acid product from the synthetic resin support at the conclusionof the preparative procedures.

It is sometimes desirable to have the coupling agents or groups presenton the dihydroxy β-lactam for reaction with the reactive groups, e.g.,hydroxyl or amino, on the synthetic resin support as illustrated above.Alternatively the coupling group may be located on the synthetic resinsupport.

A number of functional groups can be introduced into compounds of theinvention in a protected (blocked) form and subsequently de-protected toform a final, desired compound. In general, protecting groups renderchemical functionality inert to specific reaction conditions and can beappended to and removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule. The β-lactamsides,β-lactamtides, and oligo-β-lactamtides of the present invention areprepared utilizing protecting groups of diverse reactivity to allowstepwise removal when the desired functional group is needed. Some ofthe diverse types of protecting groups that are utilized in the presentinvention are acid and base labile protecting groups and protectinggroups that are removed by a fluoride source. See, e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2d edition, John Wiley & Sons,New York, 1991. For example, amino groups can be protected asphthalimido groups or as 9-fluorenylmethoxycarbonyl (FMOC) groups andcarboxyl groups can be protected as fluorenylmethyl groups.Representative hydroxyl protecting groups are described by Beaucage, etal., Tetrahedron 1992, 48, 2223. Preferred hydroxyl protecting groupsare acid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl,and trimethoxytrityl groups.

In one embodiment of the invention an oligo-β-lactamtide is preparedusing a solid support carrier. The desired β-lactam is protected at theC-4-hydroxyl position with an acid labile protecting group and at theN-1-hydroxyl position with a weak base labile protecting group or otherprotecting group that can be removed without effecting the acid labileand strong base labile protecting groups. Other functional groups thatdo not participate in the oligomerization are protected with strong baselabile protecting groups. Strong base labile protecting groups areremoved by treatment with a strong base e.g. 30% ammonium hydroxide asis used for standard DNA synthesis. The 1-N-hydroxyl group isselectively deprotected using a weak base e.g. KCN as illustrated in theexamples below. To attach the β-lactam to the solid support carrier itis necessary to couple N-1-hydroxyl of the β-lactam with a bifunctionalgroup. When the bifunctional group is attached to the β-lactam it isfurther activated with an activating group and reacted with a solidsupport using standard methods e.g. Oligonucleotide Synthesis, APractical Approach, Gait. M, J., Ed., IL: New York., 1984, Chapter 1;Masad J. Damha, nucleic acids research, 1990, 18, 3813-3821. One reagentcommonly used in DNA synthesis is succinic anhydride which is reactedwith the monomeric compound of interest and then activated with aleaving group e.g. pentafluorophenol or para nitrophenol and furtherreacted with a functional group on the solid support e.g. NH₂ on LCAACPG. A capping step is performed using a suitable capping agent e.g.acetic anhydride/lutidine/THF, and N-methyl imidazole/THF to cap anyremaining reactive sites. The acid labile protecting group on the4-hydroxyl position is removed with a dilute acid solution e.g.dichloroacetic acid or trichloroacetic acid to give the free hydroxyl atthe 4 position. The solid support carrier is washed with a suitablesolvent e.g. acetonitrile. The next step in the process is to couple thefree hydroxyl group with the phosphoramidite group of a synthoncomprising a phosphoramidite group covalently bound to a β-lactam havingthe desired protected nucleobase substituent. The resulting carrierbound dimer is oxidized from the phosphite to the phosphate to giveeither a phosphate triester or a phosphorothioate depending on theoxidizing reagent. The various types of phosphoramidite groups suitablefor use in preparing the β-lactam nucleic acids of the present inventionhave been described above.

The desired phosphoramidite/β-lactam synthon may be prepared by formingthe corresponding chloro-(2'-amino)alkoxyphosphine and thereaftercondensing this product directly with the selected β-lactam. Thereaction is carried out in an organic solvent solution of the selectedβ-lactam, preferably in the presence of a tertiary amine to take up thehydrogen chloride formed in the condensation reaction. The reactionproceeds smoothly at room temperature in a dry atmosphere and under aninert gas such as nitrogen or helium. Organic solvents useful for thisreaction include any solvent which will dissolve the reactants, such asacetonitrile, diethylether, chloroform, methylene chloride, ethylenechloride, ethyl acetate, and the like. The solution containing theproduct is separated from the precipitated hydrochloride salt of theadded tertiary amine, and can be used as such in forming β-lactamnucleic acids, or alternatively can be separated from the solvent andpurified by crystallization before further use.

Alternatively, formation of a phosphoramidite/β-lactam synthon may beachieved simply by coupling the free primary 4-hydroxyl group of theβ-lactam with a phosphoramidite group of another suitable synthon bycondensing the selected β-lactam with the phosphoramidite synthon in asuitable solvent and in the presence of an organic base such as1-H-tetrazole. Such procedures are described, e.g., in Thuong andChassignol, Tetrahedron Lett., 1987, 28, 4157; and Horn and Urdea,Tetrahedron Lett., 1986, 27, 4705. It is helpful in this reaction to useproton donors which activate the phosphoramidites. For example, acidiccompounds may be used, and are preferably mildly acidic and includeamine hydrohalide salts and nitrogen heterocyclic compounds such astetrazoles, imidazoles, nitroimidazoles, benzimidazoles, and similarnitrogen heterocyclic proton donors. The amine hydrohalide salts to beused for the protonation activation are preferably tertiary amine salts,and preferably, the hydrochloride salts, although hydrobromide,hydroiodide or hydrofluoride salts can also be used. The tertiary aminesinclude, e.g., dimethylaniline, diisopropylaniline, methylethylaniline,methyldiphenylamine, pyridine and similar teritary amines.

Once the selected β-lactam has been successfully coupled to thephosphoramidite of choice, the next step of the process is oxidizing theresulting phosphite triester to the P(V) phosphate state with oxygen orsulfur.

Such oxidation can be carried out for both phosphate (Y═O) andphosphorothioate (Y═S) structures. The oxidation can be carried outusing iodine as the oxidizing agent and under standard procedures.Oxidation can also be accomplished by reaction with peroxides such ast-butyl peroxide and benzoyl peroxide, as well as hydroperoxides. Theuse of hydrogen peroxide can lead to the formation of side products, andis, therefore, not preferred. Oxidation should be carried out beforefurther condensation of β-lactam synthons is attempted, in order toobtain the best yields. Attempts to defer oxidation until after allcondensation reactions are completed, have resulted in reduced yields ofproduct in analogous oligonucleotide preparations, due to the formationof side products.

Useful sulfurizing agents include the Beaucage reagent described ine.g., Iyer et al., J Am Chem Soc, 112, 1253-1254 (1990); and Iyer etal., J Org Chem, 55, 4693-4699 (1990); tetraethyl-thiuram disulfide asdescribed in Vu et al., Tetrahedron Lett, 32, 3005-3007 (1991);dibenzoyl tetrasulfide as described in Rao et al., Tetrahedron Lett, 33,4839-4842 (1992); di(phenylacetyl)disulfide, as described in Kamer, etal., Tetrahedron Lett, 30, 6757-6760 (1989); bis(O,O-diisopropoxyphosphinothioyl)disulfide, Wojciech J. Stec., Tetrahedron Letters, 1993,34, 5317-5320; sulfur; and sulfur in combination with ligands liketriaryl, trialkyl or triaralkyl or trialkaryl phosphines. Usefuloxidizing agents, in addition to those set out above, includeiodine/tetrahydrofuran/water/pyridine; hydrogen peroxide/water;tert-butyl hydroperoxide; or a peracid like m-chloroperbenzoic acid. Inthe case of sulfurization, the reaction is performed under anhydrousconditions with the exclusion of air, in particular oxygen; whereas, inthe case of oxidation the reaction can be performed under aqueousconditions.

Once the oxidation step has been completed the next step is to block anyunreacted sites such as hydroxyl groups that didn't react previously.This step is referred to as the capping step and it is routinelyperformed after oxidation but can be performed out of sequence dependingon the synthesis being performed. The solid support is treated with acapping reagent and washed with a suitable solvent.

More traditional blocking or capping groups can be employed, such asacid anhydrides, e.g., acetic anhydride, and arylisocyanates, and phenylisocyanate. When acetylation with acid anhydrides, e.g., aceticanhydride, is conducted in the presence of tertiary amines, especiallydi-loweralkylaminopyridines such as dimethylaminopyridine, acylationoccurs rapidly and this procedure is preferred for blocking, especiallythe N-hydroxy group of the β-lactam. The dialkylphosphite capping groupcan also be used. The resulting triester is relatively nonhydrophobicand a preferred purification involves reverse phase high performanceliquid chromatography which assures separation of the nonhydrophobicbyproduct from the product containing the hydrophobicN-O-dimethoxytrityl group.

Methods of Using β-Lactam Nucleic Acids

The β-lactam nucleic acids of the present invention may be used forresearch and in diagnostics for detection and isolation of specificnucleic acids. For example, they may be utilized in studies of enzymebiochemistry and protein-nucleic acid interactions. These and otherapplications are listed in "Oligonucleotides and Analogues: A PracticalApproach", F. Eckstein Ed., IRL Press, at pages 88-91, and in thereferences contained therein, which are hereby incorporated byreference.

As a further aspect of the invention, β-lactam nucleic acids can be usedto target RNA and ssDNA. When so used β-lactam nucleic acids are usefulas hybridization probes for the identification and purification ofnucleic acids and as therapeutic agents. Furthermore, the β-lactamnucleic acids can be modified in such a way that they can form triplehelices with dsDNA. Further reagents that bind sequence-specifically todsDNA have applications as gene targeting therapeutic agents. These areforeseen as extremely useful for treating infections and other likediseases, and may also prove effective for treatment of some geneticdiseases. The β-lactam nucleic acids of the invention can be used indiagnostics, therapeutics and as research reagents and kits. They can beused in pharmaceutical compositions by including a suitablepharmaceutically acceptable diluent or carrier. They can be used fortreating organisms having a disease characterized by the undesiredproduction of a protein. The organism should be contacted with aβ-lactam nucleic acid having a sequence that is capable of specificallyhybridizing with a strand of nucleic acid coding for the undesirableprotein. Treatments of this type can be practiced on a variety oforganisms ranging from unicellular prokaryotic and eukaryotic organismsto multicellular eukaryotic organisms. Any organism that utilizesDNA-RNA transcription or RNA-protein translation as a fundamental partof its hereditary, metabolic or cellular control is susceptible totherapeutic and/or prophylactic treatment in accordance with theinvention. Seemingly diverse organisms such as bacteria, yeast,protozoa, algae, all plants and all higher animal forms, includingwarm-blooded animals, can be treated. Further, each cell ofmulticellular eukaryotes can be treated, since they include both DNA-RNAtranscription and RNA-protein translation as integral parts of theircellular activity. Furthermore, many of the organelles (e.g.,mitochondria and chloroplasts) of eukaryotic cells also includetranscription and translation mechanisms. Thus, single cells, cellularpopulations or organelles can also be included within the definition oforganisms that can be treated with therapeutic or diagnostic β-lactamnucleic acids. As used herein, therapeutics is meant to include theeradication of a disease state, by killing an organism or by control oferratic or harmful cellular growth or expression.

The triple helix principle is believed to be the only known principle inthe art for sequence-specific recognition of dsDNA. However, triplehelix formation is largely limited to recognition ofhomopurine-homopyrimidine sequences. Strand displacement is superior totriple helix recognition in that it allows for recognition of anysequence by use of the four natural bases. Also, in strand displacementrecognition readily occurs at physiological conditions, that is, neutralpH, ambient (20°-40° C.) temperature and medium (100-150 mM) ionicstrength.

Gene targeted drugs are designed with a nucleobase sequence (containing10-20 units) complementary to the regulatory region (the promoter) ofthe target gene. Therefore, upon administration of the drug, it binds tothe promoter and blocks access thereto by RNA polymerase. Consequently,no mRNA, and thus no gene product (protein), is produced. If the targetis within a vital gene for a virus, no viable virus particles will beproduced. Alternatively, the target could be downstream from thepromoter, causing the RNA polymerase to terminate at this position, thusforming a truncated mRNA/protein which is nonfunctional.

Where the β-lactam nucleic acids of the present invention are used astherapeutic agents to treat a disease as described further above, theyare administered to a host in need of such treatment formulated in apharmaceutical composition. Such a pharmaceutical composition caninclude carriers, thickeners, diluents, buffers, preservatives, surfaceactive agents and the like in addition to the oligomer. Pharmaceuticalcompositions also can include one or more active ingredients such asantimicrobial agents, anti-inflammatory agents, anesthetiimine cs, andthe like in addition to the β-lactam nucleic acid.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically (includingtransdermally, opthalmically, vaginally, rectally, intranasally),orally, by inhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration can include sterile aqueoussolutions which also can contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with thecourse of treatment lasting from several days to several months or untila cure is effected or a diminution of disease state is achieved. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLE 1 N-1-Carboxymethylthymine

To a suspension of thymine (0.317 mole) and potassium carbonate (0.634mole) in dimethylformamide (900 ml) is added methyl bromoacetate (0.634mole) and the mixture is stirred vigorously overnight under nitrogen.The mixture is filtered, washed with ether and evaporated to dryness, invacuoto afford a colorless solid. The solid residue is treated withwater (300 ml) and 4N hydrochloric acid (12 ml), stirred for 20 minutesat 0° C., filtered and washed with water (2×100 ml). The precipitate istreated with water and 2N sodium hydroxide (60 ml), and is boiled for 10minutes. The mixture is cooled at 0° C., filtered, and then pricipitatedby the addition of 4N hydrochloric acid (70 ml) to give the titlecompound.

EXAMPLE 2 N-4-Benzoyl-N-1-carboxymethylcytosine

To a suspension of N-4-benzoylcytosine (0.317 mole) and potassiumcarbonate(0.317 mole) in dimethylformamide (900 ml), is added benzylbromoacetate (0.317 mole) and the mixture is stirred vigorouslyovernight under nitrogen. The mixture is filtered, washed with ether andevaporated to dryness, in vacuo. The solid residue is dissolved inmethanol (50 ml) and hydrogenated using Raney nickel (5 g) and hydrogenat a pressure of 40 psifor 6 hours. The reaction mixture is filtered,the catalyst washed with methanol (20 ml) and the combined fractions areconcentrated to give the title compound.

EXAMPLE 3 N-9-Carboxymethyladenine Benzyl Ester

Adenine (74 mmole) and potassium carbonate (74 mmole) are suspended inDMF (100 ml) and benzyl bromoacetate (74 mmole) in DMF (20 ml) is added.The suspension is stirred for 3 hours under nitrogen at roomtemperature, and then filtered. The solid residue is washed three timeswith DMF (25 ml), and the combined filtrate is evaporated to dryness, invacuo to give the title compound.

EXAMPLE 4 N-6-Benzoyl-N-9-carboxymethyladenine Benzyl Ester

To a stirred solution of N-9-carboxymethyladenine benzyl ester (15mmole) and pyridine (25 ml) in DMF (50 ml), in an ice bath, is added asolution of benzoic anhydride (60 mmole) in DMF (30 ml). The ice-bath isremoved and the mixture is stirred overnight. The reaction mixture isconcentratedto give the title compound as a solid.

EXAMPLE 5 N-6-Benzoyl-N-9-carboxymethyladenine

N-6-benzoyl-N-9-carboxymethyladenine benzyl ester (10 mmole) is stirredin methanol (50 ml) and hydrogenated using Raney nickel (5 g) g31 andhydrogen at a pressure of 40 psi for 6 hours. The reaction mixture isfiltered and the catalyst is washed with methanol (20 ml). The combinedfractions are concentrated to give the title compound as a solid.

EXAMPLE 6 N-9-Carboxmethylguanine Benzyl Ester

Guanine (74 mmole) and potassium carbonate (74 mmole) are suspended inDMF (100 ml) and benzyl bromoacetate (74 mmole) in DMF (20 ml) is added.The suspension is stirred for 3 hours under nitrogen at roomtemperature, and then filtered. The solid residue is washed three timeswith DMF (25 ml), and the combined filtrate is evaporated to dryness, invacuo, to give the title compound.

EXAMPLE 7 N-Benzoyl-N-9-carboxymethylguanine Benzyl Ester

To a stirred solution of N-9-carboxymethyladenine benzyl ester (15mmole) and pyridine (25 ml) in DMF (50 ml) is added a solution ofbenzoic anhydride (60 mmole) in DMF (30 ml) with ice-cooling. Theice-bath is removed and the reaction mixture is stirred overnight. Thereaction mixture is concentrated to give the title compound as a solid.

EXAMPLE 8 N-Benzoyl-N-9-carboxymethylguanine

N-Benzoyl-N-9-carboxymethyladenine benzyl ester (10 mmole) is taken upin methanol (50 ml) and hydrogenated using Raney nickel (5 g) andhydrogen ata pressure of 40 psi for 6 hours. The reaction mixture isfiltered, the catalyst washed with methanol (20 ml) and the combinedmethanol fractions are concentrated to give the title compound as asolid.

EXAMPLE 9 Preparation of Imine

O-Benzylhydroxylamine (40 mmole) is dissolved in dichloromethane (150ml), and a solution of benzyloxyacetaldehyde (40 mmole) indichloromethane (50 ml) is added slowly under nitrogen at 0° C. Thereaction mixture isstirred for 30 minutes and then 4 A molecular sieves(15 g) are added to it. After stirring for 4 hours, the reaction mixtureis filtered and concentrated to give the title compound.

EXAMPLE 101-N-Benzyloxy-3-(thymidin-1-yl)-4-benzyloxymethyl-2-azetidinone

To a stirred suspension of N-1-carboxymethylthymine (20 mmole) in DMF(50 ml) is added 2-chloro-1-methylpyridiniumiodide (20 mmole). Thereaction mixture is heated gently till the solution becomes homogenous.The reaction mixture is slowly cooled to room temperature and then asolution of imine (prepared in Example 9), (20 mmole) in DMF (20 ml) isadded slowly over a period of 20 minutes. The reaction mixture isstirred overnight and concentrated under vacuo. The crude product ispurified by flash chromatography over silica gel to give title compoundas a crystalline solid.

EXAMPLE 111-N-Benzyloxy-3-(N-benzoylcytosin-1-yl)-4-benzyloxymethyl-2-azetidinone

To a stirred suspension of N-4-benzoyl-N-1-carboxymethylcytosine (20mmole)in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole). The reaction mixture is heated gently till the solution becomeshomogenous. The reaction mixture is slowly cooled to room temperatureand then a solution of imine (prepared in Example 9) (20 mmole) in DMF(20 ml) is added slowly over a period of 20 minutes. The reactionmixture is stirred overnight and concentrated under vacuo. The crudeproduct is purified by flash chromatography over silica gel to give thethe title compound as a crystalline solid.

EXAMPLE 121-N-Benzyloxy-3-(N-benzoyladenin-9-yl)-4-benzyloxymethyl-2-azetidinone

To a stirred suspension of N-6-benzoyl-N-1-carboxymethyladenine (20mmole) in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole). The reaction mixture is heated gently till the solution becomeshomogenous. The reaction mixture is slowly cooled to room temperatureand then a solution of imine (prepared in Example 9) (20 mmole) in DMF(20 ml) is added slowly over a period of 20 minutes. The reactionmixture is stirred overnight and concentrated under vacuo. The crudeproduct is purified by flash chromatography over silica gel to give thetitle compound as a crystalline solid.

EXAMPLE 131-N-Benzyloxy-3-(N-benzoylguanin-9-yl)-4-benzyloxymethyl-2-azetidinone

To a stirred suspension of N-2-benzoyl-N-1-carboxymethylguanine (20mmole) in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole). The reaction mixture is heated gently till the solution becomeshomogenous. The reaction mixture is slowly cooled to room temperatureand then a solution of imine (prepared in Example 9) (20 mmole) in DMF(20 ml) is added slowly over a period of 20 minutes. The reactionmixture is stirred overnight and concentrated under vacuo. The crudeproduct is purified by flash chromatography over silica gel to give thetitle compound as a crystalline solid.

EXAMPLE 14 1-N-Hydroxy-3-(thymidin-1-yl)-4-hydroxymethyl-2-azetidinone

1-N-Benzyloxy-3-(thymidin-1-yl)4-benzyloxymethyl-2-azetidinone (10mmole) is taken up in methanol (50 ml) and hydrogenated using Raneynickel (5 g) and hydrogen at a pressure of 40 psi for 6 hours. Thereaction mixture is filtered, the catalyst washed with methanol (20 ml)and the combined fractions are concentrated to to give the titlecompound as a solid.

EXAMPLE 151-N-Hydroxy-3-(N-benzoylcytosin-1-yl)-4-hydroxymethyl-2-azetidinone

1-N-Benzyloxy-3-(N-benzoylcytosin-1-yl)-4-benzyloxymethyl-2-azetidinone(10mmole) is taken up in methanol (50 ml) and hydrogenated using Raneynickel (5 g) and hydrogen at a pressure of 40 psi for 6 hours. Thereaction mixture is filtered, the catalyst washed with methanol (20 ml)and the combined fractions are concentrated to afford the product as asolid.

EXAMPLE 161-N-Hydroxy-3-(N-benzoyladenin-9-yl)-4-hydroxymethyl-2-azetidinone

1-N-Benzyloxy-3-(N-benzoyladenin-9-yl)-4-benzyloxymethyl-2-azetidinone(10 mmole) is taken up in methanol (50 ml) and hydrogenated using Raneynickel(5 g) and hydrogen at a pressure of 40 psi for 6 hours. Thereaction mixture is filtered, the catalyst washed with methanol (20 ml)and the combined methanol fractions are concentrated to give the titlecompound asa solid.

EXAMPLE 171-N-Hydroxy-3-(N-benzoylguanin-9-yl)-4-hydroxymethyl-2-azetidinone

1-N-Benzyloxy-3-(N-benzoylguanin-9-yl)-4-benzyloxymethyl-2-azetidinone(10 mmole) and methanol (50 ml) is hydrogenated using Raney nickel (5 g)and hydrogen at a pressure of 40 psi for 6 hours. The reaction mixtureis filtered, the catalyst washed with methanol (20 ml) and the combinedmethanol fractions are concentrated to give the title compound as asolid.

EXAMPLE 181-N-Hydroxy-3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone

1-N-Hydroxy-3-(thymidin-1-yl)-4-hydroxymethyl-2-azetidinone (10 mmole)is dissolved in pyridine (20 ml) and 4,4'-dimethoxytrityl chloride (12mmole)is added and stirred under argon for 8 hours. The reaction mixtureis concentrated under reduced presure. The crude product is purified byflashchromatorgraphy using silica gel and ethylacetate/dichloromethane/1% triethyl amine as eluants to give the titlecompound.

EXAMPLE 191-N-Hydroxy-3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone

1-N-Hydroxy-3-(N-benzoylcytosin-1-yl)-4-hydroxymethyl-2-azetidinone (10mmole) is dissolved in pyridine (20 ml) and 4,4'-dimethoxytritylchloride (12 mmole) is added and stirred under argon for 8 hours. Thereaction mixture is concentrated under reduced pressure. The crudeproduct is purified by flash chromatorgraphy using silica gel and ethylacetate/dichloromethane/1% triethylamine as eluants to give the titlecompound.

EXAMPLE 201-N-Hydroxy-3-(N-benzoyladenin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone

1-N-Hydroxy-3-(N-benzoyladenin-9-yl)-4-hydroxymethyl-2-azetidinone (10mmole) is dissolved in pyridine (20 ml) and 4,4'-dimethoxytritylchloride (12 mmole) is added and stirred under argon for 8 hours. Thereaction mixture is concentrated under reduced pressure. The crudeproduct is purified by flash chromatorgraphy using silica gel and ethylacetate/dichloromethane/1% triethylamine as eluants to give the titlecompound.

EXAMPLE 211-N-Hydroxy-3-(N-benzoylguanin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl-2-azetidinone

1-N-Hydroxy-3-(N-benzoylguanin-9-yl)-4-hydroxymethyl-2-azetidinone (10mmole) is dissolved in pyridine (20 ml) and 4,4'-dimethoxytritylchloride (12 mmole) is added and stirred under argon for 8 hours. Thereaction mixture is concentrated under reduced pressure. The crudeproduct is purified by flash chromatorgraphy using silica gel and ethylacetate/dichloromethane/1% triethylamine as eluants to give the titlecompound.

EXAMPLE 223-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite)

To a solution of1-N-hydroxy-3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethylbis(N,N-diisopropyl)phosphoramidite (18 mmole) in acetonitrile (20 ml).The reaction mixture is stirred under argon at room temperature for 2hours. The reaction mixture is diluted with ethylacetate (75 ml), washedwith dilute sodium hydrogen carbonate (20 ml) and brine, then dried andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethyl acetate/dichloromethane as eluants to givethe title compound as a solid.

EXAMPLE 233-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite)

To a solution of1-N-hydroxy-3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(12 mmole) in acetonitrile (30 ml) is addeddiisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethylbis(N,N-diisopropyl)-phosphoramidite (18 mmole) in acetonitrile (20 ml).The reaction mixture is stirred under argon at room temperature for 2hours. The reaction mixture is diluted with ethylacetate (75 ml), washedwith dilute sodium hydrogen carbonate (20 ml) and brine then dried andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethyl acetate/dichloromethane as eluants to givethe title compound as a solid.

EXAMPLE 243-(N-benzoyladenin-9-yl)-4-(4,4'-dimethoxytrityloxymethy)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite)

To a solution of1-N-hydroxy-3-(N-benzoyladenin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethylbis(N,N-diisopropyl)phosphoramidite (18 mmole) in acetonitrile (20 ml).The reaction mixture is stirred under argon at room temperature for 2hours. The reaction mixture is diluted with ethylacetate (75 ml), washedwith dilute sodium hydrogen carbonate (20 ml) and brine then dried andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethyl acetate/dichloromethane as eluants to to givethe title compound as a solid.

EXAMPLE 253-(N-benzoylguanin-9-yl)-4-(4,4'-dimethoxytrityloxymethy)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite)

To a solution of1-N-hydroxy-3-(N-benzoylguanin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethyl bis(N,N-diisopropyl)-phosphoramidite (18 mmole) in acetonitrile (20 ml).The reaction mixture is stirred under argon at room temperature for 2hours. The reaction mixture is diluted with ethylacetate (75 ml), washedwith dilute sodium hydrogen carbonate (20 ml) and brine then dried andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethyl acetate/dichloromethane as eluants to givethe title compound as a solid.

EXAMPLE 26 Preparation of 2-azetidinone-1-N-O-succinate and2-azetidinonederivatized Controlled Pore Glass

To a stirred solution of1-N-hydroxy-3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(3.0 mmole) in anhydrous pyridine (10 ml) containing4-dimethylaminopyridine (0.180 g, 1.5 mmole) is added succinic anhydride(0.24 g, 2.4 mmole) in portions over 30 minutes. After stirringovernight,the reaction mixture is concentrated under reduced pressure toafford a gum. Toluene (30 ml) is added and co-evaporated. Thisco-evaporation is repeated twice (2×30 ml) with toluene. The residue isdissolved in dichloromethane (25 ml) and washed with ice-cold, 10%aqueous, citric acidand water and then dried. The organic extract isconcentrated to give a foam which is precipitated at room temperatureinto rapidly stirring hexane (300 ml). The mixture is centrifuged, theclear supernatant decanted and the powder dried under vacuum.

The succinylated 2-azetidinone is dissolved in dioxane (10 ml).Anhydrous pyridine (1 ml) and p-nitrophenol (0.2 g) are added followedby dicyclohexylcarbodiimide (0.6 g, 2.5 mmole). Stirring is continuedfor 3 hours. The dicyclohexylurea formed is filtered and the filtrate isadded to controlled pore glass (5 g) suspended in dimethylformamide (5ml). Triethylamine (2 ml) is added and briefly shaken. After leaving itovernight, the CPG is filtered, washed with methanol, ether and airdried.

EXAMPLE 27 Synthesis of 5-bL(GACT)-1 Phosphorothioate Tetramer

1-N-Hydroxy-3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(2 mmole) bonded to CPG (controlled pore glass) through an esterlinkageis taken in a glass reactor, and a dichloromethane solution of 2%dichloroacetic acid (volume/volume) is added to deprotect the 5-hydroxylgroup. The product is washed with acetonitrile. Then, a 0.2M solution of3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added andreacted at roomtemperature for 5 minutes. This sulfurization step is repeated one moretime for 5 minutes. The support is washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap any unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution of3-(N-benzoyladeninyl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl group. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution of3-(N-benzoylguanin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added,and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support iswashed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8). Then N-methyl imidazole/THF is added to cap the unreacted5-hydroxyl group. The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% ammoniumhydroxide solution for 2×90 minutes at room temperature. The aqueoussolution is filtered, concentrated under reduced pressure to give aphosphorothioate tetramer of 5-bL(GACT)-1.

EXAMPLE 28 Synthesis of 5-bL(GACT)-1 Phosphodiester Tetramer

1-N-Hydroxy-3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(2 mmole) bonded to CPG (controlled pore glass) through an esterlinkageis placed in a glass reactor, and a solution of 2% dichloroaceticacid in dichloromethane (volume/volume) is added to deprotect the5-hydroxy group.The product is washed with acetonitrile. A 0.2M solutionof3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile and a 0.05Msolution of tert-butyl hydroperoxide in acetonitrile is added andreacted at room temperature for 5 minutes. This oxidation step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8),and N-methyl imidazole/THF is added to cap any unreacted5-hydroxyl groups.The product is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoyladenin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoroamidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added and reacted at roomtemperature for5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of tert-butyl hydroperoxide in acetonitrile is addedand reactedat room temperature for 5 minutes. This oxidation step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap any unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoylguaninyl-9-)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added,and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of tert-butyl hydroperoxide in acetonitrile isadded and reacted at room temperature for 5 minutes. This oxidation stepis repeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to capany unreacted5-hydroxyl groups. The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% ammoniumhydroxide solution for 2×90 minutes at room temperature. The aqueoussolution is filtered and concentrated under reduced pressure to give aphosphorothioate tetramer of 5-bL(GACT)-1.

EXAMPLE 29 Preparation of Fully Protected TT Dimer

To a solution of1-N-acetoxy-3-(thymidin-1-)yl-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone(5.0 mmole) and 1-H tetrazole (4.0 mmole) in acetonitrile (60 ml) isadded3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) (6.0 mmole) in 40 ml acetonitrile. Thereaction mixture is stirred at room temperature under argon for 0.5hour. A solution of 3H-1,2-benzodithiol-3-one 1,1-dioxide (5.0 g, 25mmole) in acetonitrile is added rapidly with vigorous stirring.Thereaction mixture is stirred at room temperature for 20 minutes. Thereaction mixture is then filtered and concentrated. The crude product ispurified by flash chromatography on silica gel using ethylacetate/hexane with 1% triethylamine to give the dimer.

EXAMPLE 30 Preparation of the 5-HO-TT Dimer

The fully protected phosphorothioate TT dimer (1 mmol) is dissolved indichloromethane (50 ml) and 3% dichloroacetic acid in dichloromethane(v/v) (20 ml) is added and the mixture is stirred for 15 minutes. Thereaction mixture is concentrated and purified by flash chromatography onsilica gel using ethyl acetate/hexane with 1% triethylamine.

EXAMPLE 31 Preparation of Fully Protected CTT Phosphorothioate trimer

To a solution of 5-HO-TT phosphorothioate dimer (5.0 mmole) and 1-Htetrazole (5.0 mmole) in acetonitrile (60 ml) is added3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) (6.0 mmol) in acetonitrile (40 ml). Thereaction mixture is stirred at room temperature under argon for 0.5hour. A solution of 3H-1,2-benzodithiol-3-one 1,1-dioxide (25 mmol) inaceto nitrile is added rapidly with vigorous stirring. The reactionmixture is stirred at room temperature for 20 minutes. The reactionmixture is then filtered and concentrated. The crudeproduct is purifiedby flash chromatography on silica gel using ethyl acetate/hexane with 1%triethylamine.

EXAMPLE 32 Synthesis of 5'-d(GAC)-bL(GACT)-d(CTT)-3'-phosphorothioateDNA/LNA Mixed Sequence

5'-O-Dimethoxytritylthymidine bonded to CPG (controlled pore glass)throughan ester linkage is placed in a glass reactor, and a solution of2% dichloroacetic acid in dichloromethane (volume/volume) is added todeprotect the 5-hydroxyl groups. The product is washed withdichloromethane and then with acetonitrile. A 0.2M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl groups.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethy)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added andreacted at roomtemperature for 5 minutes. This sulfurization step is repeated one moretime for 5 minutes. The support is washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(thymidin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidin-one-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxy groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoylcytosin-1-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added andreacted at roomtemperature for 5 minutes. This sulfurization step is repeated one moretime for 5 minutes. The support is washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoyladenin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added andreacted at roomtemperature for 5 minutes. This sulfurization step is repeated one moretime for 5 minutes. The support is washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8),and N-methylimidazole/THF is added to cap the unreacted 5-hydroxyl groups.Theproduct is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution of3-(N-benzoylguanin-9-yl)-4-(4,4'-dimethoxytrityloxymethyl)-2-azetidinone-1-N-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added,and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support iswashed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to capthe unreacted5-hydroxyl groups. The product is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloeomethane (volume/volume)is added to deprotect the 5-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution ofN-4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl groups. Theproduct is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5'-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution ofN-6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8),and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

A solution of 2% dichloroacetic acid in dichloromethane (volume/volume)is added to deprotect the 5'-hydroxyl groups. The product is washed withacetonitrile. A 0.2M solution ofN-2-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl groups. Theproduct is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 2×90 minutes at room temperature. Theaqueoussolution is filtered and concentrated under reduced pressure togive the phosphorothioate decamer 5'-d(GAC)-bL(GACT)-d(CTT)-3'.

EXAMPLE 33 1-tert-Butyldimethylsilyloxy-3-hydroxypropane

To a suspension of sodium hydride (0.1 mole) in anhydroustetrahydrofuran (200 ml) cooled to 0° C. with stirring under anatmosphere of nitrogen is added a solution of 1,3-dihydroxypropane (0.1mole) in tetrahydrofuran (150 ml) slowly over a period of 1 hour. Then asolution of tert-butyldimethylsilyl chloride (0.1 mole) intetrahydrofuran (150 ml)is added slowly over a period of 1 hour.Stirring is continued for 6 hours.The mixture is then diluted with ethylacetate (400 ml), washed with water (100 ml), brine (75 ml), dried andconcentrated to afford the title compound.

EXAMPLE 34 3-tert-butyldimethylsilyloxypropionaldehyde

To a solution of anhydrous dimethylsulfoxide (0.05 mole) in anhydrousmethylene chloride (100 ml) cooled to 0° C. under nitrogen with stirringis added a solution of oxalyl chloride (0.05 mole) in anhydrousmethylene chloride (100 ml). After stirring for 15 minutes, a solutionof 1-tert-Butyldimethylsilyloxy-3-hydroxypropane (0.04 mole) inmethylene chloride (150 ml) is added over a period of 30 minutes. Thenthe reaction is quenched by adding triethyl amine (1 mole) slowly. Thereaction mixtureis diluted with ethyl acetate (200 ml), washed withwater (75 ml), brine (75 ml), dried and concentrated. The product isused as such in the subsequent condensation step.

EXAMPLE 35 2-Acetoxy-1-aminoethane

To a stirred solution of 2-aminoethanol (0.1 mole) in tetrahydrofuran(100 ml) is added distilled acetic anhydride (0.1 mole). The reactionmixture is stirred at room temperature for 12 hours and thenconcentrated to afford the title ocompound.

EXAMPLE 36 Preparation of Imine

2-Acetoxy-1-aminoethane (40 mmole) is dissolved in dichloromethane (150ml), and a solution of 3-tert-butyldimethylsilyloxypropionaldehyde (40mmole) in dichloromethane (50 ml) is added slowly under nitrogen at 0°C. The reaction mixture is stirred for 30 minutes and then 4 A molecularsieves (15 g) are added to it. After stirring for 4 hours, the reactionmixture is flitered and concentrated to give the imine.

EXAMPLE 37 N-1-Carboxyethylthymine

To a suspension of thymine (0.317 mole) and potassium carbonate (0.634mole) in dimethylformamide (900 ml) is added methyl bromopropionate(0.634mole) and the mixture is stirred vigorously overnight under anatmosphere of nitrogen. The mixture is filtered, washed with ether andevaporated to dryness in vacuo. The solid residue is treated with water(300 ml) and 4N hydrochloric acid (12 ml), stirred for 20 minutes at 0°C., filtered and washed with water (2×100 ml). The precipitate istreated with water and 2N sodium hydroxide (60 ml), and is boiled for 10minutes. The mixture is cooled at 0° C., filtered, and the titlecompound is precipitated by the addition of 4N hydrochloric acid (70 ml)to afford, after filtration, the title compound.

EXAMPLE 38 N-4-Benzoyl-N-1-carboxyethylcytosine

To a suspension of N-4-benzoylcytosine (0.317 mole) and potassiumcarbonate(0.317 mole) in dimethylformamide (900 ml) is added benzylbromopropionate (0.317 mole) and the mixture is stirred vigorouslyovernight under nitrogen. The mixture is filtered, washed with ether andevaporated to dryness, in vacuo. The solid residue is dissolved inmethanol (50 ml) and hydrogenated using Raney nickel (5 g) under 40 psipressure of hydrogen for 6 hours. The reaction mixture is filtered, thecatalyst washed with methanol (20 ml) and the combined ractions areconcentrated to afford the title compound.

EXAMPLE 39 N-9-Carboxyethyladenine Benzyl Ester

Adenine (74 mmole) and potassium carbonate (74 mmole) are uspended inDMF (100 ml) and benzyl bromopropionate (74 mmole) in DMF (20 ml) isadded. The suspension is stirred for 3 hours under nitrogen at roomtemperature, and then filtered. The solid residue is washed three timeswith DMF (25 ml), and the combined filtrate is evaporated to dryness, invacuo, to givethe title compound.

EXAMPLE 40 N-6-Benzoyl-N-9-carboxyethyladenine Benzyl Ester

To a stirred solution of N-9-carboxyethyladenine benzyl ester (15 mmole)and pyridine (25 ml) in DMF (50 ml) is added a solution of benzoicanhydride (60 mmole) in DMF (30 ml) with ice-cooling. The ice-bath isremoved and the mixture is stirred overnight. The reaction mixture isconcentrated to afford the tltle compound.

EXAMPLE 41 N-6-Benzoyl-N-9-carboxyethyladenine

N-6-benzoyl-N-9-carboxyethyladenine benzyl ester (10 mmole) is taken upin methanol (50 ml) and hydrogenated using Raney nickel (5 g) under 40psi pressure of hydrogen for 6 hours. The reaction mixture is filtered,the catalyst washed with methanol (20 ml) and the combined fractions areconcentrated to afford the title compound.

EXAMPLE 42 N-9-Carboxethylguanine Benzyl Ester

Guanine (74 mmole) and potassium carbonate (74 mmole) are suspended inDMF (100 ml) and benzyl bromopropionate (74 mmole) in DMF (20 ml) isadded. The suspension is stirred for 3 hours under nitrogen at roomtemperature, and then filtered. The solid residue is washed three timeswith DMF (25 ml), and the combined filtrate is evaporated to dryness, invacuo.

EXAMPLE 43 N-2-Benzoyl-N-9-carboxyethylguanine Benzyl Ester

To a stirred solution of N-9-carboxyethyladenine benzyl ester (15 mmole)and pyridine (25 ml) in DMF (50 ml) is added a solution of benzoicanhydride (60 mmole) in DMF (30 ml) with ice-cooling. The ice-bath isremoved and stirred overnight. The reaction mixture is concentrated toafford the title compound.

EXAMPLE 44 N-2-Benzoyl-N-9-carboxyethylguanine

N-2-Benzoyl-N-9-carboxyethyladenine benzyl ester (10 mmole) is taken upin methanol (50 ml) and hydrogenated using Raney nickel (5 g) under 40psi pressure of hydrogen for 6 hours. The reaction mixture is filtered,the catalyst washed with methanol (20 ml) and the combined fractions areconcentrated to afford the title compound.

EXAMPLE 451-N-(2-acetoxy)ethyl-3-(thymidin-1-yl)methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone

To a stirred suspension of N-1-carboxyethylthymine (20 mmole) in DMF (50ml) is added 2-chloro-1-methylpyridiniumiodide (20 mmole) and heatedgently until the solution becomes homogenous. The reaction mixture isslowly cooled to room temperature and then a solution of imine, preparedusing the procedure of Example 36, by the condensation of3-tert-butyldimethylsilyloxypropionaldehyde and 2-acetoxy-1-aminoethane(20 mmole), in DMF (20 ml) is added slowly over a period of 20 minutes.The reaction mixture is stirred overnight and concentrated under reducedpressure. The crude product is purified by flash chromatography oversilica gel to give the title compound.

EXAMPLE 461-N-(2-Acetoxy)ethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone

To a stirred suspension of N-4-benzoyl-N-1-carboxyethylcytosine (20mmole) in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole) and heated gently until the solution becomes homogenous. Thereaction mixture is slowly cooled to room temperature and then asolution of imine, prepared using the procedure of Example 36, by thecondensation of 3-tert-butyldimethylsilyloxypropionaldehyde and2-acetoxy-1-aminoethane (20 mmole) in DMF (20 ml) is added slowly over aperiod of 20 minutes. Thereaction mixture is stirred overnight andconcentrated under reduced pressure. The crude material is purified byflash chromatography over silica gel to give the title compound.

EXAMPLE 471-N-(2-acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)-methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone

To a stirred suspension of N-6-benzoyl-N-1-carboxyethyladenine (20mmole) in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole) and the mixture is heated gently until the solution becomeshomogenous. The reaction mixture is slowly cooled to room temperatureand then a solution of imine, prepared using the procedure of Example36, by the condensation of 3-tert-butyldimethylsilyloxy propionaldehydeand 2-acetoxy-1-aminoethane (20 mmole) in DMF (20 ml) is added slowlyover a period of 20 minutes. The reaction mixture is stirred overnightand concentrated under reduced pressure. The crude material is purifiedby flash chromatography over silica gel to give the title compound.

EXAMPLE 481-N-(2-acetoxy)ethyl-3-(2-N-benzoylguanin-9-yl)-methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone

To a stirred suspension of N-2-benzoyl-N-1-carboxyethylguanine (20mmole) in DMF (50 ml) is added 2-chloro-1-methylpyridiniumiodide (20mmole) and the mixture is heated gently until the solution becomeshomogenous. The reaction mixture is slowly cooled to room temperatureand then a solution of imine, prepared using the procedure of Example36, by the condensation of 3-tert-butyldimethylsilyloxy propionaldehydeand 2-acetoxy-1-aminoethane (20 mmole) in DMF (20 ml) is added slowlyover a period of 20 minutes. The reaction mixture is stirred overnightand concentrated under vacuo. The crude product is purified by flashchromatography over silica gel to afford the product as a crystallinesolid.

EXAMPLE 491-N-(2-acetoxy)ethyl-3-(thymidin-1-yl)methyl-4-hydroxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(thymidin-1-yl)methyl-4-(-2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone(prepared as per the procedure of Example 46), (10 mmole) is taken up intetrahydrofuran (50 ml) and tetrabutylammonium fluoride (25 mmole) isadded at room temperature and stirred for 2 hours. The reaction mixtureis filtered,and concentrated to give the title compound.

EXAMPLE 501-N-(2-acetoxy)ethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-hydroxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(4-N-benzoylcytosinyl)methyl-4-(-2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone(10 mmole) is taken up in tetrahydrofurane (50 ml) andtetrabutylammonium fluoride (25 mmole) is added at room temperature andstirred for 2 hours. The reaction mixture is filtered and concentratedto give the the title compound.

EXAMPLE 511-N-(2-acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-hydroxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone(10 mmole) is taken up in tetrahydrofuran (50 ml) and tetrabutylammoniumfluoride (25 mmole) is added at room temperature and stirred for 2hours. The reaction mixture is filtered and concentrated to give thetitle compound.

EXAMPLE 521-N-(2-Acetoxy)ethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-hydroxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-(2-tert-butyldimethylsilyloxy)ethyl-2-azetidinone(10 mmole) is taken up in tetrahydrofurane (50 ml) andtetrabutylammonium fluoride (25 mmole) is added at room temperature andstirred for 2 hours. The reaction mixture is filtered and concentratedto give the title compound.

EXAMPLE 531-N-(2-Acetoxy)ethyl-3-thymidin-1-ylmethyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(thymidin-1-yl)methyl-4-hydroxy-ethyl-2-azetidinone(10 mmole) is taken up in dichloromethane (50 ml) and dimethoxytritylchloride (25 mmole) and pyridine (50 mmole) are added at roomtemperature.The mixture is stirred for 12 hours. The reaction mixture isfiltered and concentrated. The crude material is purified by silica gelflash column chromatography to give the title compound.

EXAMPLE 541-N-(2-Acetoxy)ethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in dichloromethane (50 ml) and dimethoxytritylchloride (25 mmole) and pyridine (50 mmole) are added at roomtemperature and stirred for 12 hours. The reaction mixture is filteredand concentrated. The crude material is purified by silica gel flashcolumn chromatography to give the title compound.

EXAMPLE 551-N-(2-Acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-hydroxyethyl-2-azetidinone (10mmole) is taken up in dichloromethane (50 ml) and dimethoxytritylchloride(25 mmole) and pyridine (50 mmole) are added at room temperatureand stirred for 12 hours. The reaction mixture is filtered andconcentrated. The crude material is purified by silica gel flash columnchromatography to give the title compound.

EXAMPLE 561-N-(2-Acetoxy)ethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-acetoxy)ethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in dichloromethane (50 ml) and dimethoxytritylchloride (25 mmole) and pyridine (50 mmole) are added at roomtemperature and stirred for 12 hours. The reaction mixture is filteredand concentrated. The crude material is purified by silica gel flashcolumn chromatography to give the title compound.

EXAMPLE 571-N-2-Hydroxyethyl-3-(thymidin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(thymidin-1-yl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in methanol (50 ml) and potassium cyanide (15mmole) is added at room temperature and stirred for 12 hours. Thereactionmixture is filtered and concentrated. The crude material ispurified by silica gel flash column chromatography to give the titlecompound.

EXAMPLE 581-N-2-Hydroxyethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-diethoxytrityloxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in methanol 50 ml) and potassium cyanide (15mmole) is added at room emperature and the mixture is stirred for 12hours. The reaction mixture is filtered and concentrated. The crudematerial is purified by silica gel flash column chromatography to givethetitle compound.

EXAMPLE 591-N-2-Hydroxyethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in methanol (50 ml) and potassium cyanide (15mmole) is added at room temperature and stirred for 12 hours. Thereaction mixture is filtered and concentrated. The crude material ispurified by silica gel flash column chromatography to give the titlecompound.

EXAMPLE 60 1-N-2-Hydroxyethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone

1-N-(2-Acetoxy)ethyl-3-(2-N-benzoylguaninyl)methyl-4-hydroxyethyl-2-azetidinone(10 mmole) is taken up in methanol (50 ml) and potassium cyanide (15mmole) is added at room temperature and the mixture is stirred for 12hours. The reaction mixture is filtered and concentrated. The crudematerial is purified by silica gel flash column chromatography to givethetitle compound.

EXAMPLE 613-(Thymidin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropylphosphoroamidite)

To a solution of1-N-2-hydroxyethyl-3-(thymidin-1yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethyl bisN,N-diisopropyl!-phosphoramidite (18 mmole) in acetonitrile (20 ml). Thereaction mixture is stirred under an atmosphere of argon at roomtemperature for 2 hours. The reaction mixture is diluted withethylacetate(75 ml), washed with dilute sodium hydrogen carbonate (20ml) and then brine. The ethyl acetate layer is dried and concentratedunder reduced pressure. The crude product is purified by silica gelflash column chromatography using ethyl acetate/dichloromethane aseluants to give the title compound.

EXAMPLE 623-(4-N-Benzoylcytosin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoramidite)

To a solution of1-N-2-hydroxyethyl-3-(4-N-benzoylcytosin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone(12 mole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethyl bisN,N-diisopropyl!phosphoroamidite (18 mmole) in acetonitrile (20 ml). Thereaction mixture is stirred under an atmosphere of argon at roomtemperature for 2 hours. The reaction mixture is diluted withethylacetate(75 ml), washed with dilute sodium hydrogen carbonate (20ml) and then brine. The ethyl acetate layer is dried and concentratedunder reduced pressure. The crude product is purified by silica gelflash column chromatography using ethyl acetate/dichloromethane aseluants to give the title compound.

EXAMPLE 633-(6-N-Benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoroamidite)

To a solution of1-N-2-hydroxyethyl-3-(6-N-benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethyl bisN,N-diisopropyl!phosphoramidite (18 mmole) in acetonitrile (20 ml). Thereaction mixture is stirred under an atmosphere of argon at roomtemperature for 2 hours. The reaction mixture is diluted withethylacetate(75 ml), washed with dilute sodium hydrogen carbonate (20ml) and then brine. The ethyl acetate layer is dried and concentratedunder reduced pressure. The crude product is purified by silica gelflash column chromatography using ethyl acetate/dichloromethane aseluants to give the title compound.

EXAMPLE 643-(2-N-Benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoramidite)

To a solution of1-N-2-hydroxyethyl-3-(2-N-benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone(12 mmole) in acetonitrile (30 ml) is added diisopropylammoniumtetrazolide (12 mmole) followed by 2-cyanoethyl bisN,N-diisopropyl!phosphoramidite (18 mmole) in acetonitrile (20 ml). Thereaction mixture is stirred under an atmosphere of argon at roomtemperature for 2 hours. The reaction mixture is diluted withethylacetate(75 ml), washed with dilute sodium hydrogen carbonate (20ml) and then brine. The ethyl acetate layer is dried and concentratedunder reduced pressure. The crude product is purified by silica gelflash column chromatography using ethyl acetate/dichloromethane aseluants to give the title compound.

EXAMPLE 65 Synthesis of bL(GACT) Phosphorothioate Tetramer

1-N-Hydroxyethyl-3-(thymidin-1-yl)methyl-4-(dimethoxytrityloxyethyl-2-azetidinone(2 mmole) bonded to CPG (controlled pore glass) through an ester linkageis transferred to a glass reactor, and a dichloromethane solution of 2%dichloroacetic acid (volume/volume) is added to deprotect the 4-ethylhydroxyl group. The solid support and bound material is washed withacetonitrile. A 0.2M solution of3-(4-N-benzoylcytosin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support iswashed with acetonitrile and thena solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to capany unreacted 4-ethyl hydroxyl groups. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 4-ethyl hydroxyl group. The product is washedwith acetonitrile. A 0.2M solution of3-(6-N-benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support iswashed with acetonitrile and thena solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to capany unreacted 4-ethyl hydroxyl groups. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 4-ethyl hydroxyl group. The product is washedwith acetonitrile. A 0.2M solution of3-(2-N-benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added,and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support iswashed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to capany unreacted 4-ethylhydroxyl groups. The product is washed with acetonitrile.

The carrier containing the compound is treated twice with 30% ammoniumhydroxide solution for 90 minutes at room temperature. The aqueoussolution is filtered and concentrated under reduced pressure to give thephosphorothioate tetramer bL(GACT).

EXAMPLE 66 Synthesis of bL(GACT) Phosphodiester Tetramer

1-N-Hydroxyethyl-3-(thymidin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone(2 mmole) bonded to CPG (controlled pore glass) through an ester linkageis transferred to a glass reactor and a dichloromethane solution of 2%dichloroacetic acid (volume/volume) is added to deprotect the 4-ethylhydroxyl group. The product is washed with acetonitrile. A 0.2M solutionof 3-(4-N-benzoylcytosin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of 1H-tetrazole inacetonitrile is added and reacted at room temperature for 5 minutes. Theproduct is washed with acetonitrile, and then a 0.05M solution oftert-butyl hydroperoxide in acetonitrile is added and reacted at roomtemperature for 5 minutes. This oxidation step is repeated one more timefor 5 minutes. The support is washed with acetonitrile and then asolutionof acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap any unreacted 4-ethyl hydroxyl groups. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 4-ethyl hydroxyl group. The product is washedwith acetonitrile. A 0.2M solution of3-(6-N-benzoyladenin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of tert-butyl hydroperoxide in acetonitrile is added andreacted at room temperature for 5 minutes. This oxidation step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to capany unreacted 4-ethylhydroxyl groups. The product is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 4-ethyl hydroxyl group. The product is washedwith acetonitrile. Then, a 0.2M solution of3-(2-N-benzoylguanin-1-yl)methyl-4-dimethoxytrityloxyethyl-2-azetidinone-1-N-ethyl-O-(2-cyanoethylN,N-diisopropyl phosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added,and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of tert-butyl hydroperoxide in acetonitrile isadded and reacted at room temperature for 5 minutes. This oxidation stepis repeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to capany unreacted 4-ethylhydroxyl groups. The product is washed with acetonitrile.

The carrier containing the compound is treated twice with a 30% ammoniumhydroxide solution for 90 minutes at room temperature. The aqueoussolution is filtered and concentrated under reduced pressure to give thephosphorothioate tetramer bL(GACT).

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 1    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GACGACTCTT10    __________________________________________________________________________

What is claimed is:
 1. A compound having the structure: ##STR5## whereinB₁ and each B_(m) independently are a naturally occurring nucleobase, ora non-naturally occurring nucleobase;A₁ and each A_(m), independentlyare (CR₆ R₇)_(x) where R₆ and R₇ are independently selected from thegroup consisting of hydrogen, (C₂ -C₆)alkyl, aryl, aralkyl, heteroaryl,hydroxy, (C₁ -C₆)alkoxy, (C₁ -C₆)alkylthio, NR₃ R₄ and SR₅, where eachof R₃ and R₄ is independently selected from the group consisting ofhydrogen, (C₁ -C₄)alkyl, alkoxy, or alkylthio-substituted (C₁ -C₄)alkyl,alkoxy, alkylthio and amino; and R₅ is hydrogen, (C₁ -C₆)alkyl,hydroxy-, alkoxy-, or alkylthio-substituted (C₁ -C₆)alkyl, or R₆ and R₇taken together complete an alicyclic system; _(x) is an integer from 1to 10, and can be 0 only when B₁ and each B_(m) are not hydrogen orhydroxyl; E₁ and E₂ independently, are H, a hydroxyl protecting group,an activated solid support, a conjugate group, a reporter group, apolyethylene glycol, an alkyl, an oligonucleotide, a phosphate, aphosphite, an activated phosphate, or an activated phosphite; Z isselected from OH, SH, CH₃, and NR₁ R₂ ; R₁ and each R₂, independently,are H, C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂ -C₁₀ alkynyl, C₄ -C₇ carbocyloalkyl or alkenyl, a heterocycle, an ether having 2 to 10 carbon atomsand 1 to 4 oxygen or sulfur atoms, a polyalkyl glycol, or C₇ -C₁₄aralkyl; Y is selected from oxygen and sulfur; n is an integer from 1 to60; e₁ and each e_(m), independently are 0 or an integer from 1 to 6;and b₁ and each b_(m), independently are 0 or an integer from 1 to
 6. 2.The compound of claim 1 wherein B₁ and each B_(m) are, independently, anucleobase.
 3. The compound of claim 1 wherein B₁ and each B_(m) are,independently, a naturally occurring nucleobase.
 4. The compound ofclaim 1 wherein n is from 1 to about
 40. 5. The compound of claim 1wherein n is from 1 to about
 20. 6. The compound of claim 1 wherein n isfrom about 6 to about
 18. 7. The compound of claim 1 wherein E₁ ishydrogen or a hydroxyl protecting group.
 8. The compound of claim 1wherein E₂ is hydrogen or a hydroxyl protecting group.
 9. The compoundof claim 1 wherein e₁ and each e_(m) independently are an integer from 1to
 3. 10. The compound of claim 1 wherein b₁ and each b_(m)independently are an integer from 1 to
 3. 11. The compound of claim 1wherein _(x) is 0.